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A NEW SAPONIN FROM DEERlNGlA AMARANTHOIDES O.P. SATI,* S. BAHUGUNA,S . UNIYAL, DepartnMlt of Chemistry, Unimsity of Garhal, Srinagar, GarhaI246174, lndza J. SAKAKIBARA, T. KAIYA,and A . NAKAMURA Faculty of Phamueutical Srienca, Tanabe-Dwi,Mizuho-Ku Nagoya City Uniwrsity, Nagoya 467, Japan ABSTRACT.-A new triterpenoid saponin, 3-O-[a-t-rhamnopyranosyl ( 1 ~ 3 ) - 8 - ~ glucuronopyranosyl]-28-0-I&D-xylopyranosl(1~2)-~~-glucopyranosyl]-3~hydmxyolean12en-28-oace (31has been isolated together with two known saponins, 3-O-la-~-rhamnopyranosyl (1~~)-~-~-glucuronopyranosyl)-3~-hydro~olean-12-en-28-oic acid [l] and ~-O-[U-Lrhamnopyranosyl ( 1~3)-~-~-glucuronopyranosyl)-28-O-l~-~glucopyranosyll-3~-hydroxyolean-12-en-oate 121, from the fruits of Dwingia amaranthoides.
Deeringia amarantboids L. Merrill (Amaranthaceae) grows in the Western Himalayas throughout the area between 1000 and 4000 feet (1). It has been reported to possess anticancer (2)and spermicidal activity (3). A thorough survey of the literature showed that there is no report of any chemical work in this genus. Herein we report a new oleanolicacid-based saponin along with two known saponins.
RESULTS AND DISCUSSION The EtOH extract of the fruits of D . amarantboides, on repeated cc, afforded three compounds which exhibited positive color tests for triterpenoid saponins. Compound 1 in fabms (negative ion) displayed a molecular ion at mlz 777 [M - H]-. The peaks at m/z 63 1 [M -
H - 1461- and 455 [ M - H - 1461761- arose by the loss of a terminal methylpentose and a methylpentosyl hexose unit, respectively. Compounds 2 and 3 in fabms displayed molecular ions at mlz 939 [M- HI- and 107 1 [M- HIindicating the molecular weights to be 940 and 1072, respectively. Compound 3 exhibited peaks at m/z 939 [M - H 132)- and 925 {M- H - 146)- by the loss of terminal pentose and methylpenrose, respectively. The simultaneous loss of a methylpentose and a pentose unit was concluded from the ion recorded at mlz 793. The peak at m h 631 was explained by the loss ofa methylpentose, a pentose, and a hexose unit from the molecular ion. Acid hydrolysis of 1-3 furnished oleanolic acid, D-glucuronic acid, and L-
HOOC H0-q
HO
I
OH 1 R=H 2 R=B-D-glucopyranosyl 3 R=B-D-xylopyranosyl(1~2)-~-~glucopyranosyl
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rhamnose; in addition 2 afforded D-glucose and 3 gave D-glUCOSe and D-xylose. Compounds 1-3 were treated with CH2N2 and hydrolyzed to afford the methylester of oleanolic acid from 1 and oleanolic acid from 2 and 3. This confirmed that in compounds 2 and 3 the -COOH group was not free and glycosylation was at C-3 and C-28. The methylester of compounds 1-3 on NaBH4 reduction resulted in the conversion of the -COOMe group to C H 2 0 H forming the glucosyl derivatives. On permethylation (4) followed by hydrolysis, these glucosyl derivatives of 1-3 furnished 2,4,6tri-O-methyl-D-ghcose and 2,3,4-tri0-methyl-L-rhamnose, in addition 2 gave 2,3,4,6-tetra-O-methyl-~-glucose and 3 gave 2,3,4-tri-O-methyl-D-xylose and 3,4,6-tri-O-methyl-~-glucose. 3,4,6-Tri-O-methyl-~-glucose gave a positive color test with Wallenfel's reagent (5) showing a free hydroxyl group at C-2. Alkaline hydrolysis of 2 and 3 with K2C0, provided a prosapogenol, which was found to be identical to compound 1 (mmp, co-tlc, and co-ir). The partial acidic hydrolysis of 3 furnished prosapogenols PS,, PS,, PS,, and PS,, in addition to oleanolic acid. PS, and PS, were identical to compounds 1 and 2,respectively. PS, on acidic hydrolysis afforded D-glucose and glucuronic acid, while PS, gave D-glucose and Dxylose. Permethylated PS4 on methanolysis followed by hydrolysis afforded 2,3,4tri-0-methyl-D-xylose and 3,4,6-tri-0methyl-D-glucose. The types of linkages at the glycosidic bonds were confirmed by the 'H- and "C-nmr spectra. In the "C-nmr spectra of 1,the C-3 of glucuronic acid was observed at 6 83.9, revealing deshielding of ca. 6 ppm for this carbon resonance in comparison to the reported values for methyl-0-P-D-glucoside (6) as a result of glycosidation at C-3. The anomeric signals of the D-glucuronic acid and Lrhamnose were observed at 6 5.23 (d, J = 7 Hz) and 6 4.98 (s), respectively, in
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the 'H-nmr. Thus, 1 was characterized as 3-O-[a-~-rhamnopyranosyl( 1*3)-PD-glucuronopyranosyl]-3 f3-hydroxyolean- 12-en-28-oic acid. ',C nmr of 2 indicated that the inner glucuronic acid was substituted at C-3 (6 83.6 ppm, revealing deshielding of ca. 6 ppm for this carbon resonance) with a terminal rhamnose. 1H nmr showed anomeric signals at 6 5.79 (d, J z 8 . 5 Hz), 5.22 (d, J = 6 . 5 Hz), and 4.88 (s) showing the presence of Plinked glucuronic acid and D-glucose and a-linked rhamnose. Thus, 2 was characterized as 3-O-(a-~-rhamnopyranosyl ( 1~3)-P-D-glucuronopyranosyl]28-O-[P-~-glucopyranosyl)-3P-hydroxyolean- 12-en-28-oate. Methyl ethers of 1 and 2 have also been reported for the first time from the aerial part ofZexmza buphthalmifIoa (8). C-3 of glucuronic acid and C-2 of glucose appeared at 6 83.9 and 6 78.1, respectively, in the "C-nmr of 3,revealing deshielding ca. 6 pprn and ca. 5 pprn for these carbon resonances in comparison to the reported values (6). Anomeric signals in the 'H nmr appeared at 6 6.32 (d, J = 7 Hz), 6.28 (d, J = 8 Hz), 4.91 (d, J = 8 Hz), and 4.26 (br s). Thus, 3 was characterized as 3-O-[a-~-rhamnopyranosyl ( 1*3)-~-D-g~ucuronopyranosy1}-28-0-[P-D-xylopyranosyl( 1-2)P-~-glucopyranosyl]-3P-hydroxyolean12-en-28-oate. EXPERIMENTAL GENERAL EXPERIMENTAL PROCEDURES.Mp's were recorded with a Boetius microscopic apparatus, fabms JEOL, JMS-HX- 110 instrument in the negative and positive ion mode using MeOH and glycerol as solvent. 'H-nmr spectra were recorded at 400 MHz using C,D,N, and I3 C-nmr spectra were recorded at 100 MHz using CD30D, with TMS as an internal standard. Cc was performed on Si gel 6OG (Merck). The spots on tlc were visualized by spraying with 10% aqueous H 2 W 4 followed by heating. Pc was carried out on Whatman paper no. 1 using the descending mode and visualized with aniline hydrogen phthalate. The following chromatographic solvents were used: (A) petroleum ether (6040")Me,CO (l:l), (B) CHCl,-MeOH (60:40), (C) C,&-Me2CO (9: l), (D) n-BuOH-HOAc-H,O
468
Journal of Natural Products
(4: 1:5), (E) n-BuOH-EtOH-H,O (5:1:4), (F) CHCI,3-MeOH-H,O (65~40: IO), and (G) C6H6EtOAc (8:2). ISOLATION.-The fruits of D. amaranthides were collected from Romdhar (Tehri Garhwal) in July and authenticated by the Department of Botany, University of Garhwal Srinagar. Voucher specimen is preserved in the Ethnobotanical Herbarium of the Department of Botany, University of Garhwal. The air-dried material (4 kg) was defatted with solvent A in a Soxhlet apparatus. The solvent-free mass (3 kg) was exhaustively extracted with 95% EtOH until the extracts became colorless. The concentrated mass was partitioned between n-BuOH-H,O (1: 1) (4 X 500 ml). The n-BuOH extract, after concentration under reduced pressure, was dissolved in MeOH (50 ml) and precipitated in dry Me,CO (anhydrous K2C0,3), to yield a complex mixture of saponins which on repeated cc (solvent B) afforded compounds 1 (10 g), 2 (12 g), and 3 (9g). COMPOUND l.-Colorless needles (10 g) from MeOH: mp 215-220"; ir rnax (CsI) cm-' 3400 (OH), 1690; fabms m/z 799, 777, 631, 455; 'H nmr 0.70, 0.72, 0.81, 0.82, 0.84, 0.96, 1.04 (each s, 7 X tert-Me group of aglycone), 6 5.16 ( l H , br s of H-12 of aglycone), 6 5.23 ( l H , d, J = 7 Hz), 4.98 ( l H , br s) [anomeric protons] ppm; 'k nmr (C-14-30 of aglycone) 39.7, 28.7, 90.9, 38.9, 57.0, 17.8, 33.4, 40.1, 48.2, 37.8, 24.5, 123.6, 145.1, 42.7,28.6, 23.9,47.6,42.9,47.5, 31.5, 34.8, 34.0, 27.7, 17.0, 15.9, 17.7, 26.4, 178.07, 34.7, 23.9; rharnnopyranosyl 102.4 (C-l), 72.0 (C-2). 72.3 (C-3). 73.8 (C-4), 69.5 (C-5), 19.2 (C-6); glucuronopyranosyl 106.4 (C- 1). 76.2 (C2), 83.9 (C-3), 72.1 (C-4), 78.1 (C-5), 178.0 (C-
6). COMPOUND 2.--Crystallized from MeOH (12 g): mp 260-265"; ir max (CsI) cm-' 3400 (OH), 1690;fabmsm/z939,793,777,631,455; 'H nmr 0.72, 0.82, 0.96, 0.97, 1.01, 1.24, 1.30(eachs, 7 X ten-Megroup), 5.45(brsofH12 ofaglycone), 5.79 ( l H , d , J = 8 . 5 Hz), 5.22 (d,J = 6.5 Hz), 4.88 (s) [anomeric protons) ppm; "C nmr (C-14-30 of aglycone) 39.7, 28.6, 91.1, 37.8, 56.9, 17.8, 33.06, 40.0, 47.1, 37.8, 24.4, 123.7, 144.7, 42.8, 28.6, 23.98, 47.9,42.8,47.9, 31.4, 34.9, 34.8, 28.7, 17.0, 15.9, 17.7, 26.4, 178.0, 33.9, 23.9;glucuronopyranosyl 106.5 (C-l), 76.2 (C-2). 83.6 6 - 3 ) . 72.1 (C-4), 78.1 (C-5), 178.9 (C-6); rharnnopyranosyl 102.6 (C-l), 72.0 (C-2), 72.3 (C-3). 73.8 (C-4), 69.5 (C-5), 19.3 (C-6), glucopyranoSYI 95.7 (C-1). 73.8 (C-2), 77.1 (C-3), 71.1 (C4), 78.1 (C-5), 62.8 (C-6). COMPOUND3.4rystallized from MeOH (9 9): mp 27&274"; ir rnax (CsI) cm- 3400 (OH), 1680; 'H nmr 0.81, 0.89, 0.92, 0.99, 1.08,
'
Wol. 5 3 , No. 2
1.26, 1.29 (each s, 7 X ten-Me group), 5.11 ( l H , brs, H-l2ofaglycone), 6.32(1H, d , J = 7 Hz), 6.28 ( l H , d,J= 8 Hz), 4.91 (d,J= 8 Hz), 4.26 (br s) [anomeric protons] ppm; I3C nmr (C1 4 - 3 0 of aglycone) 39.8, 28.6, 91.1, 37.8, 56.8, 17.8, 33.0, 40.0, 47.1, 37.8, 24.4, 123.7, 144.7, 42.8, 28.6, 23.98, 47.9, 42.8, 47.9, 31.4, 37.8, 34.8,28.7, 17.0, 15.9, 17.7, 26.4, 178.0, 33.9, 23.9; glucuronopyranosyl 106.5 (C- l), 72.5 (C-2). 83.9 (C-3), 7 1.1 (CA), 76.9 (C-5), 177.9 (C-6); rhamnopyranosyl 102.6 (C- l), 72.0 (C-2). 72.3 (C-3), 73.8 (C-4), 69.5 (C-5), 19.3 (C-6); glucopyranosyl 99.7 (C-l), 79.2 (C-2), 78.3 (C-3), 71.4 (CA), 78.0 (C-5), 64.0 (C-6); xylopyranosyl98.7 (C-l), 74.07 (C2), 75.9 (C-3). 7 1.8 (C-4), 65.9 (C-5). ACIDICHYDROLYSIS OF COMPOUND 1.Compound 1 (25 mg) was hydrolyzed with 2 M HCIdioxane (1: 1) (3 h) on a boiling H 2 0 bath to afford the aglycone, crystallized from MeOH ( 5 ml) as colorless needles: mp 3 1&3 15". identified as oleanolic acid by direct comparison (mmp, cotlc, and co-ir) with an authentic sample: eims (MI+ 456, 441,411, 248, 208, 192. The neutralized (Ag2C03) and concentrated hydrolysate showed the presence of Dglucuronic acid and L-rhamnose (pc, solvent D, Rfvalues 0.15 and 0.37, respectively). The estimation of sugars was performed using colorimeteric methods (7).
METHYLATION.4mpOund 1 (250 mg) was dissolved in MeOH ( 5 ml) and was treated with excess ethereal CH,N2. The whole reaction mixture was allowed to stand for 12 h. Removal of the solvent under reduced pressure gave 1 methyl ester (220 mg). N&H4 REDUCTION OF 1 METHYL ESTER.The methyl ester of 1 (100 mg) was dissolved in dry Et,O (4 ml). NaBH, (500 mg) was added, and the mixture was ice-cooled, stirred for 20 hat room temperature, and evaporated in vacuo. The residue was diluted with an excess of H 2 0 and extracted with n-BuOH. The n-BuOH layer was washed, dried, and concentrated. It afforded a reduced glycoside of 1 (75 mg), ir max (GI)cm-' 3400 (OH), no COOMe. PERMETHYLATION OF THE REDUCED GLYCO-
solution of the reduced glycoside of 1 (200 mg) in MeOH was treated with dry DMSO (0.4 ml), dry t-BuONa (40 mg), finely powdered dry NaOH (10 mg), and Me1 (0.3 ml). The reaction mixture was stirred at mom temperature for 1 h. The solution was poured into iceH,O and extracted with Et,O. The E t 2 0 layer was washed with a saturated NaCl solution, dried, and evaporated. The product obtained was purified by cc (SiO,, solvent C) to hmish the methylated reduced glycoside of 1 (190 mg). SIDE OF 1.-A
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Sati et al. : New Saponin
METHANOLYSIS FOLLOWED BY HYDROLYSIS OF T H E METHYLATED REDUCED GLYCOSIDE OF 1.-A solution of the methylated reduced glycoside (50 mg) in 7% HCVdry MeOH (1 ml) was treated under reflux for 3 h and acid-hydrolyzed with 6% H,S04. T h e reaction mixture was neutralized with Ag2C03 powder and filtered. The mother liquor furnished 2,4,6-tri-O-methylDglucose and 2,3,4-tti-O-methyl-~-rhamnose (RG values 0.76 and 1.01, respectively) which were identified with authentic samples on pc (solvent E). ACIDICHYDROLYSIS OF COMPOUNDS 2 AND 3 . 4 o m p o u n d s 2 and 3 (25 mg each) were separately hydrolyzed with 2 M HCI-dioxane (1: l) (5 ml) for 3 has usual to afford an aglycone identified as oleanolic acid by its mp, co-tlc, and co-ir with an authentic sample. T h e neutralized (Ag,CO,) aqueous hydrolysates of 2 and 3on pc (solvent D) showed the presence of Dglucuronic acid, Dglucose, and Lrhamnose (R, O. 15, O. 18, 0.37, respectively). Compound 3 showed Dxylose (Rf0.28) also.
469
xylose, and 3,4,6-tri-O-methyl-~glucose (Wallenfels positive) from the permethyl ether of reduced 3. PARTIALHYDROLYSIS OF COMPOUND 3.Compound 3 (200 mg) in 1 M HCI-n-BuOH (1:l) (50 ml) was heated at 70” for 4 h. The nBuOH layer was washed with H,O and evaporated to dryness in vacuo. The residue after cc (SiO,, 15 g, solvent C) yielded four prosapogenols tentatively named as PSI (40 mg), PS, (45 mg), PS, (45 mg), and PS4 (35 mg), in addition to oleanolic acid (20 mg). PS, and PS, were found identical to compounds 1and 2, respectively (mmp and co-tlc). HYDROLYSISOF P S , . - O n acidic hydrolysis with 3% H2S04, PSI (10 mg) afforded oleanolic acid and Dglucuronic acid and Dglucose. PS,on alkaline hydrolysis gave D-glucose. NaBH, REDUCTION AND PERMETHYLATION PS,.-PS, was treated with NaBH, as above. Permethylation followed by hydrolysis afforded 2,3,4,6-tetra-O-methyl-~glucose (RG 1.00).
OF
ALKALINEHYDROYLSIS OF 2 AND 3.-Solutions of 2 and 3 (250 mg each) in MeOH (5 ml) were separately treated with 5% aqueous K,CO, (5 ml), and the whole mixture was heated under reflux for 3 h. The reaction mixture was neutralized with Dowex 50W X 8(H+) and filtered. Workup of the filtrate in the usual manner yielded a prosapogenal (210 mg), crystallized from MeOH as a colorless crystals. The prosapogenol was found to be identical to compound 1(mmp, co-tlc, and co-ir).
ACIDICHYDROLYSIS OF PS,.-PS4 (10 mg) on acidic hydrolysis afforded oleanolic acid, Dglucose, and ~ x y l o s e .
METHYLATION OF COMPOUNDS 2 AND 3.Compounds 2 and 3 (100 mg each) were separately methylated with CH,N, as described for compound 1 to furnish their methyl esters (80 and 85 mg, respectively).
The authors are thankful to JEOL Ltd., Tokyo, Japan, for fabms and University Grants Commission, New Delhi, for financial assistance.
NaBH4 REDUCTIONOF THE METHYL ESTERS OF 2 AND 3.-The methyl esters of compounds 2 and 3 (60mg each) were treated with dry Et,O and NaBH, (500 mg) as above to afford the respective methylated reduced glycosides. PERMETHYLATION FOLLOWED BY HYDROLYSIS OF THE METHYLATED REDUCED GLYCOSIDES OF 2 AND 3.-The methylated reduced glycosides of 2 and 3(50 mg each) were separately permethylated as above. The permethyl ethers thus obtained were hydrolyzed as above to furnish oleanolic acid. The mother liquors afforded 2,4,6-tri-O-methyl-~-glucose,2,3,4-tri-0methyl-L-rharnnose, and 2,3,4,&tetra-O-methylDglucose from the permethyl ether of reduced 2 and 2,4,6-tri-O-rnethyl-~glucose, 2,3,4-tri-Omethyl-L-rhamnose, 2,3,4-tri-O-methyl-D-
PERMETHYLATION OF Ps4.-=4 (20 mg) was permethylated as above, followed by acid hydrolysis, and afforded 2,3,4-tri-O-methyl-~ xylose and 3,4,6-tri-O-merhyl-~-glucose (pc, solvent D, RG 0.98 and 0.76, respectively). ACKNOWLEDGMENTS
LITERATURE CITED 1.
2. 3.
4. 5.
6. 7. 8.
A.E. Osmaston, “A Forest Flora for Kumaon,” M / S Bishan Singh, Mahendra Pal Singh, Dehra Dun, 1926, p. 425. J.L. Hartwell, Lloydia, 30,379 (1967). B.S. Setty, V.P. Kamboj, and N.M. Khanna, Indian J . Exp. Biol., 1 5 , 231 (1977). I. Ciucanu and F. Kerek, Carbohydr. Res., 1 3 1 , 209 (1984). K. Wallenfel, Natmissenschafen, 37, 49 1 (1950). S. Soe, Y.Tornita, K. Tori, and Y. Yoshimura, J. Am. C h . Sor., 1 0 0 , 3 3 3 1 (1978). S.B. Mishra and V.K. Mohan Rao, J . Sri. Ind. Res., 1 9 , 173 (1960). C.D. Schteingart and A.B. Pomilio, Phytm-hemistry, 23, 2907 (1984).
Received 2 May I989
